The Analysis Of The Quality And Yield Of DNA Extracted From Blood Cell After Long-term Cryopreservation

Objective:In genome research,the source of many cases is relatively diffused,blood samples collected cannot always be handled immediately,so these specimens need to be preserved in low temperature in biobank for months or even a few years,after the samples reached a required number,conduct a centralized extraction of DNA.The feasibility of these blood samples after long-term(over 10 years)cryopreservation is worth discussing.10 years ago,our biobank preserved DNA in the form of blood cells isolated from Ethylene Diamine Tetraacetic Acid tripotassium salt(EDTA-3K)anticoagulant blood,and drew the upper plasma for frozen storage at the same time.The method makes full use of the blood samples,reduces the blood collection of patients,and can reduce artificial operation in the middle.This article aims to investigate the quality and yield of DNA extracted from blood cell after long-term(over 10 years)storage at-80?.Methods:The 150 blood cell samples enrolled in the study were selected from the Renal Biobank of National Clinical Research Center of Kidney Diseases,which were isolated from 3ml anticoagulant blood having been preserved for over 10 years.All specimens were not through freeze and thaw cycle.Extraction of DNA was performed on automatic nucleic acid extraction instrument offered by PerkinElmer and Hamilton company,and its matching kit(Chemagic DNA Blood Kit,CMG-1073).Using magnetic beads method to extract DNA.The DNA eluting solution was 10 mM Tris-HCl pH 8.0,and the volume was 800ul.The A260/A280,A260/A230 and concentration of DNA were measured by ultraviolet spectrophotometry Nanodrop 2000(Thermo Scientific),and the yield of DNA was calculated according to the concentration and dissolution volume.The integrity of DNA was measured by agarose gel electrophoresis.If there is a bright band in the electrophoresis lane,it is suggested that there is contamination of protein.If there is a small band to in the forefront DNA electrophoresis band,indicating the contamination of RNA.If the strips are diffuse and trailing,suggesting that DNA is degraded.The A260/A280,A260/A230 and yield of DNA extracted from samples were all showed with mean and standard deviation.SPSS23.0 was used to analyze the correlation between the number of white blood cells when the samples were collected and the yield of DNA.Results:Of the DNA extracted from 150 blood cell samples,the mean value of A260/A280 is 1.84,which is in the range of 1.7?1.9,indicating extracted DNA has high purity.The mean value of A260/A230 is 2.27,which is greater than 2.0,indicating that the extracted DNA is less polluted;The average yield of DNA is 187.57?g,which is greater than 10?g,indicating that the yield of extracted DNA is sufficient for genomics research.Agarose gel electrophoresis showed that all DNA bands were relatively concentrated without dispersion distribution,and DNA molecular weight larger than 20kb.Among 150 DNA samples,there are 134 DNA 1.7?A260/A280?1.9,accounting for 89.3%,120 DNA A260/A230?2.0,accounting for 80%,115 DNA 1.7?A260/A280?1.9 and A260/A230?2.0,accounting for 76.7%.The yield of 143 DNA?10?g,accounting for 95.3%.Of which,there are 132 DNA yield?l0?g and 1.7?A260/A280?1.9,accounting for 88%.Further,114 DNA yield?10?g,1.7?A260/A280?1.9,and A260/A230?2.0,accounting for 76%.The effect of white blood cells on DNA yield was not observed.Conclusions:DNA extracted from blood cell samples that were preserved for over 10 years by magnetic beads method.And its quality and yield mostly can meet the requirements of the downstream experiment.Moreover,the researcher at our center has used the blood cell samples of our biobank to extract DNA for Genome-wide Association Study(GWAS)of diabetic nephropathy(the article is to be published),and these samples had been preserved over 10 years.This study suggests the quality and yield of DNA extracted from blood cell samples can reach a high level in such a way of saving and extraction,and provide a reference for other researchers to use samples preserved over 10 years for the following molecular research and genomics research.